This invention relates an innovative method for quantitatively separating cells, chemicals, proteins, and other ligands, or other particles, using multistage magnetically and/or electromagnetically assisted separation technology (xe2x80x9cMAGSEPxe2x80x9d). MAGSEP is extremely well suited to immunological research and analysis, pharmaceutical delivery, research and processing and other biomedical applications. Cell separation problems associated with clinical, animal, and plant research can be address using MAGSEP technology.
Almost all prior art in this field can be classified as magnetic filtration, that is, non-magnetic particles are separated from magnetic particles irrespective of their degree of magnetization. For example, Miltenyi et al., teaches that cells labeled with magnetic particles (paramagnetic, superparamagnetic or ferromagnetic) are trapped in a static tube or a flowing channel by a strong magnetic field gradient that causes them to be attracted to said tube or channel wall. Non-magnetic particles are sedimented or convected away, leaving magnetic particles captive until released from the field and collected at a later time. In U.S. Pat. No. 5,053,344, Zborowsky applies the term xe2x80x9cmagnetapheresisxe2x80x9dxe2x80x94magnetic stopping, to a similar process. Liberti et al., in U.S. Pat. No. 4,795,698 teach that thin ferromagnetic pole pieces extending into a suspension of magnetic particles will attract them, and only the magnetic particles, to said pole pieces; non-magnetic particles are convected or sedimented away, the field is switched off releasing the trapped particles into suspension where they are collected as purified cells. In a chromatography-like approach, Ugelstad teaches that high field gradients can be established around beaded ferromagnetic media and fibres, thereby trapping cells labeled with magnetic particles. Other embodiments of these magnetic filtration devices have been patented previously as set forth in U.S. Pat. Nos. 4,795,698 and 5,053,344. All of these teach a similar, simple binary separation of magnetic from non-magnetic particles, and they utilize high-gradient magnetic fields.
Prior art that is closer to the field of the invention has been presented by Powers et al., who teach that a low-gradient magnetic field applied to a horizontally flowing suspension in a channel can trap magnetically labeled cells dynamically and hence potentially according to their level of magnetization by the adsorption of magnetic particles. This method has only been applied to binary separations, however. Winoto-Morbach et al. introduced the concept of xe2x80x9cmagnetophoretic mobilityxe2x80x9d implying an intrinsic parameter whereby particles could be separated according to their speed of migration in a magnetic field gradient. Mobility is the ratio of the velocity to the driving force. In an embodiment that exploits this concept, Zborowsky et al. in U.S. Pat. No. 5,968,820, measured magnetophoretic mobilities and in U.S. Pat. No. 5,974,901 teaches that a controlled laminar flow of a suspension of particles between large permanent magnet pole pieces results in the deflection of particles according to their magnetophoretic mobility. Said deflection can be exploited as a means of recovering particles according to their mobilities, or degree of magnetization. Reddy, et. al. (1995) and Zborowski, et al. (1995) have developed analytical methods for directly evaluating the magnetization of different magnetic particle types.
Competing alternative preparative technologies consist of different types of separation processes, including electrophoresis and centrifugation. Electrophoresis involves separating materials by passing them through an electric field with separation occurring based on the attractions of the cells to one particular charge, whether positive or negative. Many of the manufacturers in this market are dedicated solely to the manufacturing of electrophoresis equipment. A centrifuge separates cells and other materials by inertial force. Heavier material is forced outward while lighter material remains on the top of the solution. This process may be beneficial when the cells separated can handle that kind of force and are able to separate based solely on size and/or density. This technique can be especially damaging to a cell, due to the high forces imposed when the unit propels cells into a container wall.
In U.S. Pat. No. 5,974,901, Zborowski et al. teach a method in which a nearly constant force field, e.g. magnetic, is applied in a region that contains cells that are caused to migrate in the force field. By capturing a series of microscope images in the force field, particle (cell) velocities can be measured and, through software, a histogram of velocities that indicate the degree of magnetization of the particles can be produced when the force field is a magnetic force field. One application of this method is the measurement of magnetophoretic mobility, the ratio of particle velocity to the applied force field, from which additional physical and chemical information about the particle can be derived. The present invention is distinguished from the Zborowski et al reference in that while Zborowski analyzes particles on the basis of a distribution of magnetic properties, the instant invention provides a means to capture them on the basis of said properties, collecting and separating particles on the basis of their magnetophoretic mobility and is not limited to the collection of merely analytical data as taught by the Zborowski reference.
In U.S. Pat. No. 5,968,820, Zborowski et al. teach a method in which a mixture of biological cells upon whose surface is affixed a number of magnetic particles in proportion to the number of receptors of interest to the researcher can be separated on that basis in a flowing stream in which they are suspended. The flowing stream flows between two magnet pole pieces, and cells within said stream are deflected toward the pole pieces at a velocity that depends on their magnetophoretic mobility and hence magnetic susceptibility and hence receptor density. The separated cells or particles are finally collected utilizing multiple outlets in fractions with each fraction containing cells having a specified range of receptor densities. Contrary to the teachings of Zborowski et al., the instant invention uses a static feed sample in a cuvette and, through the application of magnetic force, causes cells or particles to emerge from said feed cuvette with a velocity that is proportional to magnetophoretic mobility and hence magnetic susceptibility and hence receptor density.
In U.S. Pat. No. 5,053,344, Zborowski et al. teaches a system consisting of a gap between two magnetic pole pieces in which a suspension of particles is caused to flow through a thin chamber with parallel walls by gravity or some other driving means. The chamber is positioned so as to allow the particles suspended in the flowing stream to experience a spatially graded magnetic force. The spatially graded magnetic force causes the capture of particles spatially distributed on a plane according to their magnetic susceptibility in a process traditionally termed xe2x80x9cferrographyxe2x80x9d. Subsequent to capture, some particles, especially biological cells, can be examined according to the position at which they were captured and classified, but not collected in suspension according to magnetic susceptibility and hence, if labeled with liganded magnetic particles, receptor density. This system does not separate particles collectible in suspension and therein differs from the instant invention, which is designed to accomplish such separation and collection.
Improved techniques for separating living cells and proteins are increasingly important to biotechnology because separation is frequently the limiting factor for many biological processes. In response to that need, the present invention was developed to provide a method for quantitatively separating cells, particles, ligands, proteins, and other chemical species using a magnetic and/or an electromagnetically-assisted separation process.
The instant apparatus and method of use provides an innovative method for quantitatively separating cells, proteins, or other particles, using multistage, magnetically and/or electromagnetically assisted separation technology (xe2x80x9cMAGSEPxe2x80x9d). The MAGSEP technology provides a separation technology applicable to medical, chemical, cell biology, and biotechnology processes. Moreover, the instant invention relates to a method for separating and isolating mixtures of combinatorial synthesized molecules such that a variety of products are prepared, in groups, possessing diversity in size, length, (molecular weight), and structural elements. These are then analyzed for the ability to bind specifically to an antibody, receptor, or other ligate. Such a collection may provide a ligand library containing specific ligands for any ligate even though there are a greater number of conformations available to any one sequence. This technology provides a cell biologists a tool for studying molecular recognition. Combinational chemical libraries containing known and random sequences can be surveyed for strong ligands. Such a tool provides a means of recognizing and isolating agonists, antagonists, enzyme inhibitors, virus blockers, antigens, and other pharmaceuticals.
In clinical applications utilizing a single or multistage magnetic and/or electromagnetic separator, cells that are labeled with decreasing numbers of paramagnetic beads are separated quantitatively on the basis of the extent of labeling by using magnetic fields of increasing strength. Cells with greater numbers of magnetic beads attached to their receptors will be attracted to a weak magnetic field, while cells with fewer beads will not as shown best in FIG. 1. This principle establishes the basis for separating (xe2x80x9cclassifyingxe2x80x9d) cells or other particles according to their magnetic strength, using either a rate or an equilibrium process.
One main reason that electromagnetic field-assisted methods have not been heavily employed commercially in the past is the mystique of equipment used in the field. The physics is considered too complex, but it is rather simple in fact. There is further misunderstanding about the mechanism of separation. In addition to the existence of a mystique, real physical factors also have been a deterrent to magnetic field-assisted separations. Most magnetically assisted separations that require the specific adsorption of beaded media to the separand also require some kind of flowing device for removing unwanted particles.
The multistage electromagnetic separator of the instant invention overcomes these barriers by greatly simplifying the electromagnetic field-assisted separation process. The separator does not require a stabilized matrix such as gel, paper, or density gradient. The technology does not require any forced flow of fluid for magnetic separation. The iterative transfer of fluids minimizes flows and provides a milder and more suitable environment for separating and purifying cells and proteins. The electromagnetic separator technology incorporated into the present invention also offers automatic decanting of contaminant suspensions. The unwanted cells or particles are simply left behind as by-products of the process in an opposing half chamber. Finally, the end-user of the apparatus will appreciate the added efficiency of needing to make only one buffer to complete extraction and to collect automatically separated fractions without the complications of pumping and volume measurements.
Another application of magnetic separation technology that is in its infancy is the development of neoglycoconjugates. Many cells, enzymes, and lectins possess recognition sites for specific carbohydrates (xe2x80x9clectinxe2x80x9d means xe2x80x9ccarbohydrate binding proteinxe2x80x9d). By conjugating specific carbohydrates (oligo- or polysaccharides) to the surface of magnetic beads, specific cells, enzymes or lectins can be isolated by HMGS or MACS. This represents an ideal application for MAGSEP, since different glycoconjugates can be linked to magnetic beads of different strengths, thus separating, out of a mixed population, cells that recognize glycoconjugate A on strongly magnetizable beads from those that recognize glycoconjugate B on weakly magnetizable beads. Furthermore, MAGSEP could also cause the collection of bead-free cells at the end of the separation by adding a solution of free sugars that competed for the magnetic binding sites thereby setting the magnetically captured cells free.
In addition to the above very recent innovation, needs for the separation of cells on the basis of receptor density have been identified. Research laboratories have recently used receptor number as a dependent variable in a variety of scientific applications. In endocrinology mouse leukemia cells exhibit reduced beta-adrenergic receptors, in growth regulation the number of EG. receptors is regulated by cell density in cultures which can be modulated by protamine, in virology the cell surface has limited numbers of receptors for herpes virus glycoprotein D which is required for virus entry into cells, in carcinogenesis the H-ras oncogene alters the number and type of EG.-beta receptors, in infectious diseases galanin receptor levels are coupled to pertussis toxin resistance of pancreatic cells, and a diphtheria toxin receptor-associated protein has been identified. In neurology regulation of opioid kappa receptors occurs in stimulated brain cell cultures, in nutrition mast cells lose IgE receptors in protein malnutrition, and vasoactive intestinal peptide (VIP) receptors have been discovered at high density. This relatively small sample of recent findings indicates clearly that tools for studying cells with modified receptor densities would be welcome.
Methods exist for utilizing high-magnetic-gradient technology for the specific removal of cells from the human circulation by labeling them with immunobead ligands. This is now practiced as a binary separation which might benefit from continuous separation afforded by the instant invention. The use of magnetically delivered therapeutics is another potential application for magnetic particle separation technology.
Once magnetized particles or microcapsules for delivery have been made, it is necessary to separate weakly magnetized particles from those with the highest susceptibility. Since strongly magnetized particles will be required, an important consideration is the distance between the external magnet and the delivery site and the undesirability of delivering weak particles, loaded with drug, to normal-tissue sites to produce unwanted side effects. The technology may be utilized as a means for the separation of a specified subset of T-lymphocytes for transfusion of AIDS patients, or a specified subset of islet cells for the treatment of diabetes.
The counting of prepurified cells in diagnostic tests parallels developments in flow cytometry which costs up to 100 times as much. The low cost of this technology can not be overstated: AIDS care givers in the developing world are puzzled over how to do diagnostic tests that involve flow cytometry in environments that lack flow cytometers. The instant invention utilizing a multistage electromagnetic separator solves these problems and promises to offer solutions to such global health problems.
In theory, there are no capacity limits to magnetically-assisted separation. It can be small, for diagnostic purposes, or large, for preparative applications such as cell transplants. The latter is significant since a tall magnetic column, which would be required (possibly up to 1 meter and a field greater than 1-2 Teslas) for the quantitative resolution we propose, is replaced by the staged separation cavities in a rotating disk with several modest permanent magnets and electromagnets as illustrated in FIG. 2.
The development of user-friendly devices that are capable of separating particles according to quantity of ligand on their surfaces appears to be the greatest need in improving magnetically-assisted separation devices. The magnetic separation industry has made considerable progress in this regard, but the technology to date has been limited to binary separation methods. An example would be Baxter Healthcare""s Isolex-300 Magnetic Cell Separator, which chooses stem/progenitor cells through use of monoclonal antibody (MAB)-coated magnetic beads. The stem cells are selected for reconstituting bone marrow damaged by chemical or radiation treatment. The instant MAGSEP invention represents a quantum leap in progress by finally providing a reliable method for differential separation on the basis of small differences in surface composition.
Most ligand-based (such as receptor-antibody) cell separation methods are binaryxe2x80x94all or nothing. By combining magnetic attraction, used as a rate process, with countercurrent extraction, it is now possible to use magnetic separation of cells as a quantitative technique, separating on the basis of the number of ligands bound per cell. This could be qualitative, based on the amount of ligand bound to each kind of cell, or quantitative, based on the amount of ligand bound to cells of the same type, some with high receptor content and some with low.
It is an object of the present invention to provide a method for quantitatively separating cells, proteins, or other particles, using multistage, magnetically, electromagnetically assisted separation technology, (xe2x80x9cMAGSEPxe2x80x9d).
It is an object of the instant invention to provide a method for separating and isolating mixtures of combinatorial synthesized molecules such that a variety of products are prepared, in groups, possessing diversity in size, length, (molecular weight), and structural elements which may be analyzed for the ability to bind specifically to an antibody, receptor, or other ligate, providing a means for forming a ligand library containing specific ligands for any ligate to provide a cell biologists a tool for studying molecular recognition.
It is an object of the present invention to provide a means of recognizing and isolating agonists, antagonists, enzyme inhibitors, virus blockers, antigens, and other pharmaceuticals using combinational chemical libraries containing known and random sequences.
It is a further object of the present invention to provide a method of magnetic cell and cell components sorting for plants and animals.
It is another object of the present invention to develop a plate assembly capable of incorporating at least one and preferably a multiple of magnets, electromagnetic devices, and/or combinations thereof and base support.
It is another object of the present invention to design electromagnetic hardware and drive boards capable of providing variable field strength (in the 1-1000 mT range).
It is another object of the present invention to design an indexing system for plate translation.
It is another object of the present invention to incorporate and configure the electromagnetic separator of the present invention to fit within an containment enclosure for space flight and remote applications.
It is another object of the present invention to incorporate data management and processing control system.
It is another object of the present invention to provide an electromagnet exhibiting a relatively quick change in polarity to enhance mixing.
It is another object of the present invention to provide an electromagnetic separator having a constant force and a formed flux density.
It is an object of the present invention to provide an embodiment, whereby biological cells that have on their surfaces receptors that can be bound by an antibody can be attached to magnetic particles through specific chemical ligands such as avidin, a protein that reacts with biotin, a vitamin that can be chemically bound to the antibody thereby attaching the cells to magnetic particles to be collected by the present invention.
It is another object of the present invention to select homogeneous populations of magnetic particles from heterogeneous magnetic particle populations synthesized for use in cell research applications.
It is another object of the present invention to select strong, homogeneous populations of magnetic particles for targeted drug delivery whereby magnetic microparticles are used for the parenteral delivery of targeted drugs based wherein the differentiation and selection due to the fact that magnetically weak particles are inimical to this modality.
It is another object of the present invention to utilize an embodiment wherein the translating magnet is a permanent dipole, a permanent quadrupole, or a permanent hexapole magnet, or the magnet is a dipolar, quadrupolar or circular electromagnet.
It is another object of the present invention to utilize an embodiment wherein the translating magnet is a series of fixed electromagnets of any polarity, operated in sequence so as to sweep particles into a common starting band.
It is another object of the present invention to utilize an embodiment wherein the control of the translating magnet(s) holding magnet(s) and disk transfer system is controlled by a computer and custom software.
It is another object of the present invention to utilize an embodiment wherein capture cavities and holding magnets are arrayed in a straight line or some other geometrical relationship especially including in a circle.
It is another object of the present invention to utilize an embodiment wherein more than one sample cuvette, with their translating magnets, serve the array of capture cavities.
It is another object of the present invention to utilize an embodiment wherein the invention is used to separate magnetically labeled biological cells.
It is another object of the present invention to utilize an embodiment wherein the invention is used to select homogeneous populations of magnetic microparticles for application to cell separation and other biochemical separation processes.
It is another object of the present invention to utilize an embodiment wherein the invention is used to select homogeneous subpopulations of magnetic particles for targeted drug delivery.
It is another object of the present invention to utilize an embodiment wherein the invention is used in any process in which the desired goal is the classification (separation) of magnetic particles according to magnetophoretic mobility and hence volumetric differential susceptibility.
It is another object of the present invention to utilize an embodiment wherein no translation magnet is used.
It is another object of the present invention to provide a reciprocating magnetic collector comprising magnetic particle separator in which at least one sample cuvette filled with a liquid in which particles to be separated are suspended and at least one reuseable capture cavity positioned so as to interface with the fluid in said sample cuvette.
It is another object of the present invention to provide an optical magnetocytometer comprising at least one light source, including optical elements such as lenses, filters and mirrors, and at least one light detector, including optical elements such as lenses, filters and mirrors causing particles to be sensed as they are collected into the capture cavity.
It is another object of the present invention to provide a multistage high-gradient separator in which the capture of low-susceptibility particles is facilitated by at least one polepiece of ferromagnetic metal or other magnetizable substance permanently or temporarily positioned within the capture cavity of the primary invention.
These and other objects of the present invention will be more fully understood from the following description of the invention.